Abstract/ Summary For decades, blockade of CD40-CD154 interactions following transplantation has been shown to be a highly effective means of inhibiting alloreactive T cell responses and inducing long-term survival of allografts, and under some conditions, transplantation tolerance in both murine and non-human primate models. However, the potential of this therapeutic strategy to have a transformative impact on transplantation outcomes has yet to be realized. Specifically, problems associated with the fact that anti-CD154 mAbs may cause thromboembolism by binding and cross-linking CD154 on platelets via Fc-dependent mechanisms stymied the clinical translation of CD154 blockers, and instigated the therapeutic targeting of CD40 as an alternative therapy. While these anti-CD40 reagents certainly possess the ability to significantly prolong allograft survival, none has achieved the remarkable tolerance-inducing results observed with anti-CD154 mAbs. These observations raise the possibility that blockade of CD154 vs. blockade of CD40 are actually not mechanistically equivalent. As these anti-CD40 reagents make their way through the pipeline for clinical translation in transplantation, it is imperative to determine if there is a biological explanation underlying the observed inferiority of blocking CD40 as compared to blocking CD154, in order to then devise ways to overcome it. Here, we present compelling new preliminary data revealing that CD11b is a second receptor for CD154 during alloimmunity, signaling through which is blocked by anti-CD154 reagents but not anti-CD40 reagents. Using a specific peptide antagonist, we find that CD154:CD11b interactions function locally within the allograft to enhance donor-reactive CD8+ T cell infiltration and accelerate allograft rejection. However, the mechanisms by which CD154:CD11b interactions promote T cell migration into allografts are unknown. Moreover, induction of Foxp3+ iTreg is a critical effect in anti-CD154-elicted transplantation tolerance, but our new preliminary data show iTreg are not induced via CD40 blockade or in CD40-/- recipients. Thus, we will also interrogate the role of blocking novel CD154:CD11b interactions in inducing Foxp3+ iTreg and promoting tolerance during alloimmunity. Results from the proposed experiments will illuminate novel CD154-dependent aspects of alloimmunity that are not blocked by anti-CD40 mAbs, thus filling a gap in our understanding of fundamental mechanisms of allograft rejection. The proposed experiments are also highly clinically relevant, because as anti-CD40 mAbs move through the pipeline for clinical translation, understanding the impact of CD154:CD11b interactions (that proceed unimpeded in the setting of CD40 blockade) is of the utmost importance in order to devise innovative strategies to block these interactions and thus optimize the use of CD40 blockers to facilitate transplantation tolerance in the clinic.